Studies with isolated plasma membranes indicate that agonist binding to the alpha1-adrenergic receptor may be regulated by GTP and its nonhydrolyzable analogues. To further study alpha1- receptor:effector interactions, a soluble hormone-receptor complex was prepared by incubating rat liver membranes with norepinephrine followed by detergent solubilization with digitonin. The addition of guanyl nucleotides to the solubilized receptors facilitated the release of tightly bound norepinephrine. Binding of the alpha1-adrenergic receptor antagonist, (3H)-prazosin, to the soluble preparation was utilized as a gauge of guanyl nucleotide- induced release of receptor-bound agonist. The following potency series was obtained with regard to the ability of guanyl nucleotides to facilitate (3H)-prazosin binding to the solubilized preparation: guanosine 5'-0-(3-thiotriphosphate) greater than guanylyl-beta, gamma, imidodiphosphate greater than guanosine triphosphate much greater than adenylyl-beta, gamma, imidodiphosphate. Competitive binding studies indicated that the receptor had a higher affinity for prazosin than for either the non-selective alpha antagonist phentolamine, or the alpha2- selective antagonist, yohimbine, thereby confirming the receptor's identity as the alpha1-adrenergic subtype. Photo-affinity labeling studies with the prazosin analog (125I)-aryl azidoprazosin indicated a molecular weight of approximately 90,000 for the guanyl nucleotide-sensitive alpha1-adrenergic receptor.